Gabriela Orasanu

Retinaldehyde represses adipogenesis and diet-induced obesity.

The metabolism of vitamin A and the diverse effects of its metabolites are tightly controlled by distinct retinoid-generating enzymes, retinoid-binding proteins and retinoid-activated nuclear receptors. Retinoic acid regulates differentiation and metabolism by activating the retinoic acid receptor and retinoid X receptor (RXR), indirectly influencing RXR heterodimeric partners. Retinoic acid is formed solely from retinaldehyde (Rald), which in turn is derived from vitamin A. Rald currently has no defined biologic role outside the eye. Here we show that Rald is present in rodent fat, binds retinol-binding proteins (CRBP1, RBP4), inhibits adipogenesis and suppresses peroxisome proliferator-activated receptor-γ and RXR responses. In vivo, mice lacking the Rald-catabolizing enzyme retinaldehyde dehydrogenase 1 (Raldh1) resisted diet-induced obesity and insulin resistance and showed increased energy dissipation. In ob/ob mice, administrating Rald or a Raldh inhibitor reduced fat and increased insulin sensitivity. These results identify Rald as a distinct transcriptional regulator of the metabolic responses to a high-fat diet.

The Pathologic Continuum of Diabetic Vascular Disease

Hyperglycemia can promote vascular complications by multiple mechanisms, with formation of advanced glycation end products and increased oxidative stress proposed to contribute to both macrovascular and microvascular complications. Many of the earliest pathologic responses to hyperglycemia are manifest in the vascular cells that directly encounter elevated blood glucose levels. In the macrovasculature, these include endothelial cells and vascular smooth muscle cells. In the microvasculature, these include endothelial cells, pericytes (in retinopathy), and podocytes (in renal disease). Additionally, neovascularization arising from the vasa vasorum may promote atherosclerotic plaque progression and contribute to plaque rupture, thereby interconnecting macroangiopathy and microangiopathy.

PPARs and their metabolic modulation: new mechanisms for transcriptional regulation?

Peroxisome proliferator‐activated receptors (PPARs) as ligand‐activated nuclear receptors involved in the transcriptional regulation of lipid metabolism, energy balance, inflammation, and atherosclerosis are at the intersection of key pathways involved in the pathogenesis of diabetes and cardiovascular disease. Synthetic PPAR agonists like fibrates (PPAR‐α) and thiazolidinediones (PPAR‐γ) are in therapeutic use to treat dyslipidaemia and diabetes. Despite strong encouraging in vitro, animal model, and human surrogate marker studies with these agents, recent prospective clinical cardiovascular trials have yielded mixed results, perhaps explained by concomitant drug use, study design, or a lack of efficacy of these agents on cardiovascular disease (independent of their current metabolic indications). The use of PPAR agents has also been limited by untoward effects. An alternative strategy to PPAR therapeutics is better understanding PPAR biology, the nature of natural PPAR agonists, and how these molecules are generated. Such insight might also provide valuable information about pathways that protect against the metabolic problems for which PPAR agents are currently indicated. This approach underscores the important distinction between the effects of synthetic PPAR agonists and the unequivocal biologic role of PPARs as key transcriptional regulators of metabolic and inflammatory pathways relevant to diabetes and atherosclerosis.

PPARγ in the endothelium regulates metabolic responses to high-fat diet in mice

Although endothelial dysfunction, defined as abnormal vasoreactivity, is a common early finding in individuals with type 2 diabetes, the endothelium has not been known to regulate metabolism. As PPARgamma, a transcriptional regulator of energy balance, is expressed in endothelial cells, we set out to investigate the role of endothelial cell PPARgamma in metabolism using mice that lack PPARgamma in the endothelium and BM (gammaEC/BM-KO). When gammaEC/BM-KO mice were fed a high-fat diet, they had decreased adiposity and increased insulin sensitivity compared with control mice, despite increased serum FFA and triglyceride (TG) levels. After fasting or olive oil gavage, gammaEC/BM-KO mice exhibited significant dyslipidemia and failed to respond to the FFA and TG lowering effects of the PPARgamma agonist rosiglitazone. BM transplantation studies, which reconstituted hematopoietic PPARgamma, established that these metabolic phenotypes were due to endothelial PPARgamma deficiency. We further found that the impairment in TG-rich lipoprotein metabolism in gammaEC/BM-KO mice was associated with fatty acid-mediated lipoprotein lipase inhibition and changes in a PPARgamma-regulated endothelial cell transcriptional program. Despite their metabolic improvements, high-fat diet-fed gammaEC/BM-KO mice had impaired vasoreactivity. Taken together, these data suggest that PPARgamma in the endothelium integrates metabolic and vascular responses and may contribute to the effects of PPARgamma agonists, thus expanding what endothelial function and dysfunction may entail.

Reciprocal and Coordinate Regulation of Serum Amyloid A Versus Apolipoprotein A-I and Paraoxonase-1 by Inflammation in Murine Hepatocytes

Objectives—During inflammation, the serum amyloid A (SAA) content of HDL increases, whereas apolipoprotein A-I (apoA-I) and paraoxonase-1 (PON-1) decrease. It remains unclear whether SAA physically displaces apoA-I or if these changes derive from coordinated but inverse transcriptional regulation of the HDL apolipoprotein genes. Because cytokines stimulate the hepatic expression of inflammatory markers, we investigated their role in regulating SAA, apoA-I, and PON-1 expression. Methods and Results—A cytokine mixture (tumor necrosis factor [TNF]-&agr;, interleukin [IL]-1&bgr;, and IL-6) simultaneously induced SAA and repressed apoA-I and PON-1 expression levels. These effects were partially inhibited in cells pretreated with either nuclear factor &kgr;B (NF-&kgr;B) inhibitors (pyrrolidine dithiocarbamate, SN50, and overexpression of super-repressor inhibitor &kgr;B) or after exposure to the peroxisome proliferator-activated receptor-&agr; (PPAR&agr;) ligands (WY-14643 and fenofibrate). Consistent with these findings, the basal level of SAA was increased, whereas apoA-I and PON-1 decreased in primary hepatocytes from PPAR&agr;-deficient mice as compared with wild-type mice. Moreover, neither WY-14643 nor fenofibrate had any effect on SAA, apoA-I, or PON-1 expression in the absence of PPAR&agr;. Conclusion—These results suggest that cytokines increase the expression of SAA through NF-&kgr;B transactivation, while simultaneously decreasing the expression of apoA-I and PON-1 by inhibiting PPAR&agr; activation. Inflammation may convert HDL de novo into a more proatherogenic form by coordinate but inverse transcriptional regulation in the liver, rather than by physical displacement of apoA-I by SAA.

High-Density Lipoprotein Hydrolysis by Endothelial Lipase Activates PPARα. A Candidate Mechanism for High-Density Lipoprotein-Mediated Repression of Leukocyte Adhesion

Although high-density lipoprotein (HDL) is known to inhibit endothelial adhesion molecule expression, the mechanism for this anti-inflammatory effect remains obscure. Surprisingly, we observed that HDL no longer decreased adhesion of U937 monocytoid cells to tumor necrosis factor (TNF)&agr;-stimulated human endothelial cells (EC) in the presence of the general lipase inhibitor tetrahydrolipstatin. In considering endothelial mechanisms responsible for this effect, we found that endothelial lipase (EL) overexpression in both EC and non-EL–expressing NIH/3T3 mouse embryonic fibroblasts cells significantly decreased TNF&agr;-induced VCAM1 expression and promoter activity in a manner dependent on HDL concentration and intact EL activity. Given recent evidence for lipolytic activation of peroxisome proliferator-activated receptors (PPARs)—nuclear receptors implicated in metabolism, atherosclerosis, and inflammation—we hypothesized HDL hydrolysis by EL is an endogenous endothelial mechanism for PPAR activation. In both EL-transfected NIH cells and bovine EC, HDL significantly increased PPAR ligand binding domain activation in the order PPAR-&agr;≫-&ggr;>-&dgr;. Moreover, HDL stimulation induced expression of the canonical PPAR&agr;-target gene acyl-CoA-oxidase (ACO) in a PPAR&agr;-dependent manner in ECs. Conditioned media from EL-adenovirus transfected cells but not control media exposed to HDL also activated PPAR&agr;. PPAR&agr; activation by EL was most potent with HDL as a substrate, with lesser effects on LDL and VLDL. Finally, HDL inhibited leukocyte adhesion to TNF&agr;-stimulated ECs isolated from wild-type but not PPAR&agr;-deficient mice. This data establishes HDL hydrolysis by EL as a novel, distinct natural pathway for PPAR&agr; activation and identifies a potential mechanism for HDL-mediated repression of VCAM1 expression, with significant implications for both EL and PPARs in inflammation and vascular biology.

The Peroxisome Proliferator-Activated Receptor-γ Agonist Pioglitazone Represses Inflammation in a Peroxisome Proliferator-Activated Receptor-α–Dependent Manner In Vitro and In Vivo in Mice

OBJECTIVES Our aim was to investigate if the peroxisome proliferator-activated receptor (PPAR)-gamma agonist pioglitazone modulates inflammation through PPARalpha mechanisms. BACKGROUND The thiazolidinediones (TZDs) pioglitazone and rosiglitazone are insulin-sensitizing PPARgamma agonists used to treat type 2 diabetes (T2DM). Despite evidence for TZDs limiting inflammation and atherosclerosis, questions exist regarding differential responses to TZDs. In a double-blinded, placebo-controlled 16-week trial among recently diagnosed T2DM subjects (n = 34), pioglitazone-treated subjects manifested lower triglycerides and lacked the increase in soluble vascular cell adhesion molecules (sVCAM)-1 evident in the placebo group. Previously we reported PPARalpha but not PPARgamma agonists could repress VCAM-1 expression. Since both triglyceride-lowering and VCAM-1 repression characterize PPARalpha activation, we studied pioglitazones effects via PPARalpha. METHODS Pioglitazone effects on known PPARalpha responses--ligand binding domain activation and PPARalpha target gene expression--were tested in vitro and in vivo, including in wild-type and PPARalpha-deficient cells and mice, and compared with the effects of other PPARgamma (rosiglitazone) and PPARalpha (WY14643) agonists. RESULTS Pioglitazone repressed endothelial TNFalpha-induced VCAM-1 messenger ribonucleic acid expression and promoter activity, and induced hepatic IkappaBalpha in a manner dependent on both pioglitazone exposure and PPARalpha expression. Pioglitazone also activated the PPARalpha ligand binding domain and induced PPARalpha target gene expression, with in vitro effects that were most pronounced in endothelial cells. In vivo, pioglitazone administration modulated sVCAM-1 levels and IkappaBalpha expression in wild-type but not PPARalpha-deficient mice. CONCLUSIONS Pioglitazone regulates inflammatory target genes in hepatic (IkappaBalpha) and endothelial (VCAM-1) settings in a PPARalpha-dependent manner. These data offer novel mechanisms that may underlie distinct TZD responses.

Dual Roles for Lipolysis and Oxidation in Peroxisome Proliferation-Activator Receptor Responses to Electronegative Low Density Lipoprotein

Low density lipoprotein (LDL) exists in various forms that possess unique characteristics, including particle content and metabolism. One circulating subfraction, electronegative LDL (LDL(–)), which is increased in familial hypercholesterolemia and diabetes, is implicated in accelerated atherosclerosis. Cellular responses to LDL(–) remain poorly described. Here we demonstrate that LDL(–) increases tumor necrosis factor α (TNFα)–induced inflammatory responses through NFκB and AP-1 activation with corresponding increases in vascular cell adhesion molecule-1 (VCAM1) expression. LDL receptor overexpression increased these effects. In contrast, exposing LDL(–) to the key lipolytic enzyme lipoprotein lipase (LPL) reversed these responses, inhibiting VCAM1 below levels seen with TNFα alone. LPL is known to act on lipoproteins to generate endogenous peroxisomal proliferator-activated receptor α (PPARα) ligand, thus limiting inflammation. These responses varied according to the lipoprotein substrate triglyceride content (very low density lipoprotein ≫ LDL > high density lipoprotein). The PPARα activation seen with LDL, however, was disproportionately high. We show here that MUT LDL activates PPARα to an extent proportional to its LDL(–) content. As compared with LDL(–) alone, LPL-treated LDL(–) increased PPARα activation 20-fold in either cell-based transfection or radioligand displacement assays. LPL-treated LDL(–) suppressed NFκB and AP-1 activation, increasing expression of the PPARα target gene IκBα, although only in the genetic presence of PPARα and with intact LPL hydrolysis. Mass spectrometry reveals that LPL-treatment of either LDL or LDL(–) releases hydroxy-octadecadienoic acids (HODEs), potent PPARα activators. These findings suggest LPL-mediated PPARα activation as an alternative catabolic pathway that may limit inflammatory responses to LDL(–).

VLDL hydrolysis by LPL activates PPAR-α through generation of unbound fatty acids

Recent evidence suggests that lipoproteins serve as circulating reservoirs of peroxisomal proliferator activated receptor (PPAR) ligands that are accessible through lipolysis. The present study was conducted to determine the biochemical basis of PPAR-α activation by lipolysis products and their contribution to PPAR-α function in vivo. PPAR-α activation was measured in bovine aortic endothelial cells following treatment with human plasma, VLDL lipolysis products, or oleic acid. While plasma failed to activate PPAR-α, oleic acid performed similarly to VLDL lipolysis products. Therefore, fatty acids are likely to be the PPAR-α ligands generated by VLDL lipolysis. Indeed, unbound fatty acid concentration determined PPAR-α activation regardless of fatty acid source, with PPAR-α activation occurring only at unbound fatty acid concentrations that are unachievable under physiological conditions without lipase action. In mice, a synthetic lipase inhibitor (poloxamer-407) attenuated fasting-induced changes in expression of PPAR-α target genes. Apolipoprotein CIII (apoCIII), an endogenous inhibitor of lipoprotein and hepatic lipase, regulated access to the lipoprotein pool of PPAR-α ligands, because addition of exogenous apoCIII inhibited, and removal of endogenous apoCIII potentiated, lipolytic PPAR-α activation. These data suggest that the PPAR-α response is generated by unbound fatty acids released locally by lipase activity and not by circulating plasma fatty acids.

Retinaldehyde dehydrogenase 1 coordinates hepatic gluconeogenesis and lipid metabolism.

Recent data link vitamin A and its retinoid metabolites to the regulation of adipogenesis, insulin sensitivity, and glucose homeostasis. Retinoid metabolism is tightly controlled by an enzymatic network in which retinaldehyde dehydrogenases (Aldh1-3) are the rate-limiting enzymes that convert retinaldehyde to retinoic acid. Aldh1a1-deficient mice are protected from diet-induced obesity and hence diabetes. Here we investigated whether Aldh1a1 and the retinoid axis regulate hepatic glucose and lipid metabolism independent of adiposity. The impact of Aldh1a1 and the retinoid pathway on glucose homeostasis and lipid metabolism was analyzed in hepatocytes in vitro and in chow-fed, weight-matched Aldh1a1-deficient vs. wild-type (WT) mice in vivo. Aldh1a1-deficient mice displayed significantly decreased fasting glucose concentrations compared with WT controls as a result of attenuated hepatic glucose production. Expression of key gluconeogenic enzymes as well as the activity of Forkhead box O1 was decreased in Aldh1a1-deficient vs. WT livers. In vitro, retinoid or cAMP agonist stimulation markedly induced gluconeogenesis in WT but not Aldh1a1-deficient primary hepatocytes. Aldh1a1 deficiency increased AMP-activated protein kinase α activity, decreased expression of lipogenic targets of AMP-activated protein kinase α and significantly attenuated hepatic triacylglycerol synthesis. In metabolic cage studies, lean Aldh1a1-deficient mice manifested enhanced oxygen consumption and reduced respiratory quotient vs. WT controls, consistent with increased expression of fatty acid oxidation markers in skeletal muscle. Taken together, this work establishes a role for retinoid metabolism in glucose homeostasis in vivo and for Aldh1a1 as a novel determinant of gluconeogenesis and lipid metabolism independent of adiposity.